This invention was produced in part using funds obtained through a grant from the National Institutes of Health. Consequently, the federal government has certain rights in this invention.
1. Field of the Invention
The present invention relates generally to adenoviral vectors and adenoviral gene therapy. More specifically, the present invention relates to an infectivity-enhanced conditionally replicative adenovirus.
2. Description of the Related Art
Surgery, chemotherapy and radiotherapy constitute the conventional therapies in clinical use to treat cancer. These therapies have produced a high rate of cure in early-stage cancer, but most late-stage cancers remain incurable because they cannot be resected or the dose of radiation or chemotherapy administered is limited by toxicity to normal tissues. An alternative promising approach is the transfer of genetic material to tumor or normal cells as a new therapy itself or to increase the therapeutic index of the existing conventional therapies [1]. In this regard, three main strategies have been developed to accomplish cancer gene therapy: potentiating immune responses against tumors, eliciting direct toxicity to tumors, and compensating the molecular lesions of tumor cells [2].
To achieve the high level of gene transfer required in most cancer gene therapy applications, several viral and non-viral vectors have been designed [13]. Adenoviral vectors have been used preferentially over other viral and non-viral vectors for several reasons, including infectivity of epithelial cells, high titers, in vivo stability, high levels of expression of the transgene, gene-carrying capability, expression in non-dividing cells, and lack of integration of the virus into the genome. In most of the adenoviral vectors used in cancer gene therapy, the transgene substitutes for the early 1 region (E1) of the virus. The E1 region contains the adenoviral genes expressed first in the infectious stage and controls expression of the other viral genes. The early region 3 (E3) gene codes for proteins that block a host""s immune response to viral-infected cells and is also usually deleted in vectors used for cancer gene therapy, particularly in immunopotentiating strategies.
E1-substituted, E3-deleted vectors can carry up to 8 kb of non-viral DNA, which is sufficient for most gene therapy applications. E1-substituted, E3-deleted vectors are propagated in packaging cell lines that transcomplement their E1-defectiveness, with production yields of up to 10,000 virion particles per infected cell, depending upon the transgene and its level of expression in the packaging cell. Not all of the viral particles are able to transduce cells or to replicate in the packaging cell line, so bioactivity of a particular vector has been defined as the ratio of functional particles to total particles. This bioactivity varies from 1/10 to 1/1000, depending not only upon the vector, but also upon the methods of purification and quantification [15]. The titer used is the concentration of functional particles, which can be as high as 1012 per milliliter.
One problem encountered when propagating these vectors to high titers is the recombination of vector sequences with the E1 sequences present in the packaging cell line, thereby producing replication-competent adenoviruses (RCA). This problem has been solved by using packaging cell lines where the E1 gene does not overlap with the vector sequences [16].
The current generation of adenoviral vectors are limited in their use for cancer gene therapy, primarily for three reasons: (1) the vectors are cleared by the reticuloendothelial system, (2) the vectors are immunogenic and/or (3) the vectors infect normal cells. The problem of filtration by the reticuloendothelial system cells, such as macrophages of the spleen or Kupffer cells of the liver, affects adenoviral vectors as well as other viral and non-viral vectors and limits their utility in intravascular administration [19]. The early and late viral genes that remain in E1-E3 deleted vectors may also be expressed at low, but sufficient enough levels such that the transduced cells are recognized and lysed by the activated cytotoxic T lymphocytes. Additionally, a higher viral dose must be injected to reach the entire tumor before a neutralizing immune response develops. The major limitation then becomes the amount of vector that can be safely administered, which will depend upon the capacity of the vector to affect tumor cells without affecting normal cells.
The limitations of adenoviral vectors at the level of infectivity is two-fold. On the one hand, human clinical trials with adenoviral vectors have demonstrated relatively inefficient gene transfer in vivo. This has been related to the paucity of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR), on tumor cells relative to their cell line counterparts [20-23]. On this basis, it has been proposed that gene delivery via CAR-independent pathways may be required to circumvent this key aspect of tumor biology. On the other hand, adenoviral vectors efficiently infect normal cells of many epithelia. This results in the expression of the transgene in normal tissue cells with the consequent adverse effects. This problem has been addressed by targeting adenoviral vectors to tumor cells at the level of receptor interaction and transgene transcription.
Targeting adenoviral vectors to new receptors has been achieved by using conjugates of antibodies and ligands, in which the antibody portion of the conjugate blocks the interaction of the fiber with the CAR receptor and the ligand portion provides binding for a novel receptor [20]. Receptor targeting has also been achieved b y genetic modification of the fiber [23-26]. Transcriptional targeting of adenoviral vectors has further been demonstrated using tumor-antigen promoters or tissue-specific promoters to control the expression of the transgene [27]. However, these promoters can lose their specificity when inserted in the viral genome and, depending upon the level of toxicity of the transgene, even low levels of expression can be detrimental to normal cells. Thus, for cancer gene therapy, the major issues limiting the utility of adenoviral vectors are the efficiency and specificity of the transduction.
The major limitation found in the use of adenoviral vectors in the clinical setting is the number of tumor cells that remain unaffected by the transgene. A vector that propagates specifically in tumor cells, results in lysis and subsequently allows for transduction of neighbor cells by newly produced virions will increase the number of tumor cells affected by the transgene [28]. A good replicative vector should be weakly pathogenic or non-pathogenic to humans and should be tumor-selective [29]. Efforts have been aimed at improving the safety of replication-competent adenoviruses with the goal of being able to administer much higher doses. One strategy is to transcomplement the E1 defect with an E1-expression plasmid conjugated into the vector capsid [31], which allows a single round of replication thereby producing a new E1-substituted vector with the ability of local amplification and subsequent gene transduction.
Other strategies are designed to obtain vectors that replicate continuously and whose progeny are also able to replicate, but are incapable of propagating in normal cells. In this regard, two approaches have been described that render adenovirus propagation selective for tumor cells: (1) deletions, and (2) promoter regulation [30]. Adenoviral mutants unable to inactivate p53 propagate poorly in cells expressing p53 but efficiently in tumor cells where p53 is already inactive. Based upon this strategy, an adenovirus mutant in which the E1b-55k viral protein was deleted and was unable to bind to p53 was effective in eliminating tumors in preclinical models and is in clinical trials [32]. Controlling viral replication by substituting a viral promoter, such as the E1a promoter, with a tumor associated-antigen promoter, such as the alpha-fetoprotein promoter or the prostate antigen promoter, has been demonstrated [33], and specific lysis of tumors transfected with an adenovirus vector expressing either of the above-mentioned promoters was demonstrated in murine models.
Both approaches have limitations, however. The fact that other viral proteins besides E1b 55K also interact with p53, and because p53 can be necessary for the active release of virus in the later stages of infection may affect the specificity of the vector [37,38]. Another caveat results from using E1a as the only controlled viral gene since E1a-like activity has been found in many tumor cell lines [14,40]. Furthermore, the actual specificity of the above-mentioned promoters for cancer cells, and the fact that promoters inserted in the viral genome can lose their expression specificity, are factors that hindered clinical applications of this approach [39].
Therefore, new methods are clearly needed to achieve more selective therapeutic effects of replication-competent adenoviruses. For these vectors, in parallel to what has been achieved with non-replicative vectors, modification of viral tropism could enhance tumor transduction and tumor selectivity at the level of cell entry, and in this way, realize the full potential of replicative vectors for cancer gene therapy.
The prior art is deficient in adenoviral vectors that are specific for a particular cell type (i.e., do not infect other cell types) and that replicate with high efficiency in only those particular cell types. The present invention fulfills this long-standing need and desire in the art.
Adenoviral vectors have been widely employed in cancer gene therapy. Their high titers, structural stability, broad infectivity, high levels of transgene expression, and lack of integration have contributed to the utility of this vector. In this regard, adenoviral vectors has been used to transfer a variety of genes to treat cancer such as cytokines, tumor suppresser genes, pro-drug converting genes, antisense RNAs and ribozymes to inhibit the expression of oncogenes, antiangiogenic genes, etc. Despite the promise of adenoviral vectors, results from experimental models and clinical trials have been less than optimal.
Within this context, several specific limitations have been identified. One limitation lies in the poor infectability of primary tumors due to low levels of the primary adenovirus receptor CAR. A second limitation that particularly affects the efficiency of replicative vectors is related to the lack of tumor-specific replication achieved using promoters or mutations. The present invention describes methods to increase adenovirus infectivity based upon modification of the virus tropism. The present invention demonstrates that modification of the adenovirus fiber by genetic manipulation increases infectivity of primary tumors several orders of magnitude due to CAR-independent gene transfer. In addition, selective replication in tumors is described herein, and represents a safe and effective means to lyse and transduce tumors. The present invention further describes a strategy based upon control of the expression of one or more essential early viral genes using tumor-specific promoters.
It is a goal of the present invention to improve the infectivity and specificity of conditional replicative vectors, thereby improving their therapeutic utility and efficacy.
One object of the present invention is to provide adenoviral vectors that possess enhanced infectivity to a specific cell type (i.e., that are not limited to CAR-dependent cell entry) and that replicate with high efficiency in only those cell types.
In an embodiment of the present invention, there is provided an infectivity-enhanced conditionally-replicative adenovirus. This adenovirus possesses enhanced infectivity towards a specific cell type, which is accomplished by a modification or replacement of the fiber of the adenovirus. The modification is accomplished by introducing a fiber knob domain from a different subtype of adenovirus, introducing a ligand into the HI loop of the fiber knob, or replacing the fiber with a substitute protein which presents a targeting ligand. Additionally, the adenovirus has at least one conditionally regulated early gene, such that replication of the adenovirus is limited to the specific cell type.
In yet another embodiment of the present invention, there is provided a method of enhanced-infectivity conditionally-replicative adenoviral gene therapy in an individual in need of such treatment. This method comprises the steps of: administering to an individual a therapeutic dose of an infectivity-enhanced conditionally-replicative adenovirus. This adenovirus possesses enhanced infectivity towards a specific cell type, which is accomplished by a modification or replacement of the fiber of the adenovirus. The modification is accomplished by introducing a fiber knob domain from a different subtype of adenovirus, introducing a ligand into the HI loop of the fiber knob, or replacing the fiber with a substitute protein which presents a targeting ligand. The adenovirus also has at least one conditionally regulated early gene, such that replication of the adenovirus is limited to the specific cell type.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.